CN116179663B - Method for realizing nucleic acid single-molecule fragment sequencing based on gamma-hemolysin nano pore canal contraction region - Google Patents
Method for realizing nucleic acid single-molecule fragment sequencing based on gamma-hemolysin nano pore canal contraction region Download PDFInfo
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- 239000002773 nucleotide Substances 0.000 claims description 3
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- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 3
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- ZVQOOHYFBIDMTQ-UHFFFAOYSA-N [methyl(oxido){1-[6-(trifluoromethyl)pyridin-3-yl]ethyl}-lambda(6)-sulfanylidene]cyanamide Chemical compound N#CN=S(C)(=O)C(C)C1=CC=C(C(F)(F)F)N=C1 ZVQOOHYFBIDMTQ-UHFFFAOYSA-N 0.000 abstract 1
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
Abstract
The invention discloses a method for realizing nucleic acid single-molecule fragment sequencing based on gamma-hemolysin nano pore canal contraction region, which comprises the following steps: constructing a nano patch clamp device, arranging solid holes between cis-form and trans-form detection cells of an electrolytic cell, and arranging a salt solution; attaching a phospholipid layer to the solid state holes to block the solid state holes; the gamma-hemolysin protein forms an octamer structure consisting of a vestibule and a beta-barrel structure; the connecting area is a contraction area; constructing gamma-hemolysin nano pore canal on the phospholipid layer to enable the liquid between the forward and reverse detection tanks to be communicated; applying a voltage to obtain an opening current; adding a nucleic acid single molecule aqueous solution into the cis-detection cell, and applying voltage to enable the nucleic acid single molecule to pass through the contraction zone in a fragment-type manner; measuring the blocking current; and identifying the nucleic acid single molecule according to the sequence information, and obtaining fragment sequencing information. The invention can accurately detect the dynamics characteristic of the metastable state of the double-chain, realize the high spatial resolution identification of 3 bases at the tail end of the identification code and realize the high-accuracy sequencing of the target sequence.
Description
Technical Field
The invention relates to a method for realizing nucleic acid single-molecule fragment sequencing based on a gamma-hemolysin nano pore canal contraction region.
Background
The nanopore sensing technology starts with the coulter counting concept patented in 1953 [1] . To explore further intracellular genetic information, deamer, branton and Kasianowicz et al reported for the first time that protein nanopores detect nucleotides [2] . Protein nanopores currently enable information extraction (including length) of DNA, RNA, polypeptides, and proteins [3] Sequence of [4] Modified bases [5] Etc.) and molecular conformation [6] And small molecular substances [7] Is detected. This is excellentThe sensing performance is derived from the fact that the sensing performance serves as the only channel between the two chambers of the electrolytic cell for allowing ions in the solution to pass through. And forming stable potential difference on two sides of the nano pore canal by using an external power supply, so that sample information is reflected according to the blocking current condition.
Gamma-hemolysin (gamma-hemolysin) is a protein from staphylococcus aureus. The nano pore canal has octamer beta-barrel structure and larger vestibular area than alpha-hemolysin nano pore canal [8] . In 2018, the gamma-hemolysin nanopore (HlgC-HlgB) channel was first used as a DNA structural analysis by Cherie S.tan et al, and successfully achieved the change discrimination of double-stranded DNA bases G to I [8] . The short stay of the double-chain sequence can be realized by utilizing the high sensing sensitivity and the special space size of the gamma-hemolysin nano pore canal contraction region, but the research of high-precision detection and distinguishing different double chains has not been reported yet.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for realizing single-molecule fragment sequencing of nucleic acid based on a gamma-hemolysin nano pore canal contraction region.
The technical scheme of the invention is summarized as follows:
a method for realizing single-molecule fragment sequencing of nucleic acid based on gamma-hemolysin nano-pore canal constriction region comprises the following steps: constructing a nano patch clamp device, wherein a solid hole is arranged between a cis-type detection tank and a trans-type detection tank of an electrolytic tank in the nano patch clamp device, so that the cis-type detection tank and the trans-type detection tank are communicated, and salt solution is respectively arranged in the cis-type detection tank and the trans-type detection tank; attaching a phospholipid layer to the solid-state well to block the solid-state well between the cis-detection cell and the trans-detection cell; preparing gamma-hemolysin protein, wherein the gamma-hemolysin protein is assembled by polypeptides from F and S respectively to form an octamer structure composed of vestibule and beta-barrel structures; the vestibule and the connecting area of the beta-barrel-shaped structure are contraction areas; constructing a gamma-hemolysin nano pore canal on the phospholipid layer to enable liquid between the cis-type detection cell and the trans-type detection cell to be communicated; applying a voltage to obtain an opening current; adding a nucleic acid single molecule aqueous solution into the cis-detection pool, and applying voltage to enable the nucleic acid single molecule to pass through the contraction zone from the cis-terminal fragment of the gamma-hemolysin nano pore canal; measuring a blocking current through the gamma-hemolysin nanopore; and identifying the nucleic acid single molecules according to the measured blocking current, and obtaining fragment sequencing information.
The concentration of the salt solution is 2-4M, and the solvent of the salt solution is HOAc/KOAc buffer solution with pH=5.0.
The salt is potassium chloride or sodium chloride.
The nucleic acid single molecule is a target sequence and an identification code corresponding to the target sequence, at least 2 sections of complementary hybrid molecules are formed, and the nucleic acid single molecule is of an A-type double-helix structure, and the size of the A-type double-helix structure is structurally matched with the size of a contraction zone.
The contraction region is any combination of amino acid sequences from No. 104 to No. 112 and No. 141 to No. 152 in the F polypeptide shown by one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 and amino acid sequences from No. 100 to No. 109 and No. 132 to No. 141 in the S polypeptide shown by one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.
The target sequence is first DNA or first RNA, and the identification code corresponding to the first DNA is second RNA or PNA; the identification code corresponding to the first RNA is the second DNA.
The invention has the advantages that:
the invention takes off the inherent thinking of extremely narrow nanopore direct reading and biological enzyme control speed, combines the identification code and the target sequence to form a single-double chain alternate conformation, and utilizes the gamma-hemolysin nanometer pore canal contraction zone to accurately detect the dynamics characteristics of double-chain metastable state conformations, thereby realizing the high spatial resolution identification of 3 bases at the tail end of the identification code and realizing the high-accuracy sequencing of the target sequence.
Drawings
FIG. 1 shows the complementation of the target sequence and the identification code.
FIG. 2 shows the whole process of detecting nucleic acid single molecules in a sample by using a gamma-hemolysin nanopore.
FIG. 3 shows the gamma-hemolysin nanopore opening current at a drive voltage of 100 mV.
FIG. 4 shows the electrical signal of a two-segment RNA identifier and a target sequence via at a drive voltage of 100 mV.
Detailed Description
The invention is further illustrated by the following examples.
Example 1
A method for realizing single-molecule fragment sequencing of nucleic acid based on gamma-hemolysin nano-pore canal constriction region comprises the following steps: constructing a nano patch clamp device, regulating the initial voltage to 30mV by using a temperature control instrument, and controlling the temperature to 20.0+/-0.5 ℃; an electric signal is 0pA, a solid hole (glass nano hole) with the diameter of 2 micrometers is arranged between a cis-type detection cell and a trans-type detection cell of an electrolytic cell in the nano patch clamp device, the cis-type detection cell and the trans-type detection cell are communicated (the cis-type detection cell and the trans-type detection cell are the only way for electrolyte to flow), 3M potassium chloride solution (a solution of the potassium chloride solution is 20mM HOAc/KOAc buffer solution with the pH value of 5.0 or can be any concentration potassium chloride solution or sodium chloride solution with the concentration of 2M-4M) is injected into the cis-type detection cell and the trans-type detection cell respectively by a syringe, so that the electric signal disappears. The same phenomenon is seen repeatedly for a plurality of times, which shows that the solid holes (glass nano holes) are clean and smooth and can be used for subsequent experiments;
attaching a phospholipid layer to the solid state pores:
the method comprises the steps of dissolving the powder of the di-phytic phosphatidylcholine (DPhPC) in decane to obtain a solution I with the concentration of 10mg/mL, then dripping the solution I into a saline solution in a cis detection pool to enable the final concentration of the di-phytic phosphatidylcholine to be 0.0025mg/mL, and adjusting the liquid level position through multiple times of suction by a syringe to promote the di-phytic phosphatidylcholine to pass through and attach to the glass nano-pores. When the current signal value was observed to be 0pA at the initial voltage applied, regardless of whether the salt solution level was over the solid state wells, it was shown that the phospholipid layer was successfully attached to the solid state wells. Then applying breaking air pressure of 100Pa (a value can be selected in 100-130 Pa), if the electric signal suddenly disappears after air pressure within a certain air pressure range is applied, the phospholipid layer is broken and has moderate thickness, and if the phospholipid layer is too thick, the phospholipid needs to be refilled. Blocking solid state pores between the cis detection cell and the trans detection cell; when the sampling rate is 10kHz, the noise is less than 2pA, the subsequent experiment can be started,
preparing gamma-hemolysin protein, wherein the gamma-hemolysin protein is assembled by polypeptides from F and S respectively to form an octamer structure composed of vestibule and beta-barrel structure, and the connecting region of the vestibule and beta-barrel structure is a contraction region:
a solution of class F polypeptide HlgB (SEQ ID NO.4 commercially available) and class S polypeptide HlgC (SEQ ID NO.8 commercially available) and 100mM Deoxycholate (DOC) (100 mM Tris-HCl as solvent) was mixed (HlgB/HlgC/DOC mass ratio 1:1:10), and the mixture was gradually stirred in 100mM Tris-HCl buffer (pH 8.3) to a final concentration of 6.25mM DOC and 3.5. Mu.M (a mixture of class S polypeptide HlgC and class F polypeptide HlgB). The culture was carried out at room temperature for two hours, and the monomers were removed by a 100kDa cut-off centrifugal filter (Amicon Ultra 100K apparatus) and purified to obtain gamma-hemolysin protein.
The contraction region is any combination of amino acid sequences from No. 104 to No. 112 and No. 141 to No. 152 in the F-class polypeptide shown in SEQ ID NO.4 and amino acid sequences from No. 100 to No. 109 and No. 132 to No. 141 in the S-class polypeptide shown in SEQ ID NO. 8.
Constructing gamma-hemolysin nano pore canal on the phospholipid layer
Adding 5 μg gamma-hemolysin protein into a cis-detection cell, applying a voltage of 30Pa (optionally any one of 20-40Pa and 200mV (optionally any one of 100-350 mV) to help the gamma-hemolysin protein to be embedded into a phospholipid layer, then scanning the device by positive and negative voltages, and constructing a gamma-hemolysin nano pore channel on the phospholipid layer under the action of the applied voltage to enable liquid between the cis-detection cell and the trans-detection cell to be communicated;
preparation of nucleic acid single molecules:
preparation of the first DNA of the target sequence by solid phase Synthesis (SEQ ID NO.10:5' - (A) 30 -GCAGAGTACT AACCA-(A) 15 TAACCACTATACGAT-3') and a second RNA (SEQ ID NO.11:5'-UGGUUAGUACUCUGC-3', SEQ ID NO.12:5'-AUCGUAUAGUGG UUA-3'), each sample was purified over 30 minutes after synthesis using an anion exchange HPLC purification method, while UV absorbance was monitored at 260 nm. ddH of sample at 4 DEG C 2 Dialysis in O for 36 hours to remove purified salts, followed by lyophilization. Obtaining ddH by measuring absorbance at 260nm and estimating extinction coefficient using the first order sequence 2 O re-suspends the concentration of the first DNA or second RNA molecule.
Obtaining nucleic acid single molecule blocking current signal
Adding a nucleic acid single molecule aqueous solution (the molar concentration ratio of the second RNA of the identification code to the first DNA of the target sequence is 2:1) into the cis-detection pool, forming a plurality of complementary sequences in the pool (see figure 1), wherein the nucleic acid single molecule is the target sequence and the identification code corresponding to the target sequence, forming at least 2 complementary hybrid molecules, and is of an A-type double-helix structure, the size of the A-type double-helix structure is matched with the size structure of a contraction zone, and applying voltage (60 mV applied voltage) to enable the nucleic acid single molecule to pass through the contraction zone from the cis-end fragment of the gamma-hemolysin nano pore canal (see figure 2); the segment shift process includes the first segment of complementary sequence stopping briefly and the second segment of complementary sequence contacting the nanometer pore canal shrinkage area until all the identification codes fall off.
Measuring a blocking current through the gamma-hemolysin nanopore with 20mV as a first gear, up to 200mV at each voltage regulation; until a high blocking current signal occurs;
signal collection and processing:
the obtained blocking current signal is processed by QuB software to obtain time domain frequency domain signal characteristics, the signal is further analyzed by Matlab software, data are analyzed and mapped by combining with OriginLab software, and the nucleic acid single molecules are identified according to the measured blocking current, so that fragment sequencing information is obtained (see FIG. 4). The single nucleic acid molecule is a 2-segment complementary hybrid molecule formed by the first DNA and the two RNAs.
Experiments prove that the first DNA and PNA or the first RNA and the second DNA can form complementary sequences for detecting single molecules of nucleic acid.
The amino acid sequences from No. 104 to No. 112 and No. 141 to No. 152 in the F-class polypeptides shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3 can be used for replacing the amino acid sequences from No. 104 to No. 112 and No. 141 to No. 152 in the F-class polypeptides shown in SEQ ID NO.4, and other embodiments are the same, so that a new embodiment is formed;
the 100 th to 109 th and 132 th to 141 th amino acid sequences in the S-type polypeptides shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.9 can be used for replacing the 100 th to 109 th and 132 th to 141 th amino acid sequences in the S-type polypeptides shown in SEQ ID NO.8, and the same examples are used for forming new examples.
Reference to the literature
1.Crosland-Taylor PJ:A Device for Counting Small Particles suspended in a Fluid thr ough a Tube.Nature 1953,171:37-38.
2.Volkov AG,Deamer DW:Liquid-liquid interfaces:theory and methods.In 1996,13:42-46.
3.Cao C,et al.Discrimination of oligonucleotides of different lengths with a wild-ty pe aerolysin nanopore.Nature nanotechnology 2016,11:713-718.
4.Laszlo AH,et al.Decoding long nanopore sequencing reads of natural DNA.Nature biotechnology 2014,32:829-833.
5.Wallace EV,et al.Identification of epigenetic DNA modifications with a pro-tein na nopore.Chemical communications 2010,46:8195-8197.
6.Wang Y,Guan X,Zhang S,et al.Structural-profiling of low molecular weight RNA s by nanopore trapping/translocation using Mycobacterium smegmatis porin A.Nat Commun2021,12:3368.
7.J.Ettedgui,J.J.Kasianowicz and A.Balijepalli.Single Molecule Discrimi-nation of Heteropolytungstates and Their Isomers in Solution with a Nanometer-Scale Pore.J Am Chem Soc 2016,138:7228-7231.
8.Tan CS,Fleming AM,Ren H,et al.γ-Hemolysin Nanopore Is Sensitive to Guanine-to-Inosine Substitutions in Double-Stranded DNAat the Single-Molecule Level.J Am Chem Soc 2018,140:14224-14234。
Claims (3)
1. A method for realizing single-molecule fragment sequencing of nucleic acid based on gamma-hemolysin nano-pore canal constriction region is characterized by comprising the following steps: constructing a nano patch clamp device, wherein a solid hole is arranged between a cis-type detection tank and a trans-type detection tank of an electrolytic tank in the nano patch clamp device, so that the cis-type detection tank and the trans-type detection tank are communicated, and salt solution is respectively arranged in the cis-type detection tank and the trans-type detection tank; attaching a phospholipid layer to the solid-state well to block the solid-state well between the cis-detection cell and the trans-detection cell; preparing gamma-hemolysin protein, wherein the gamma-hemolysin protein is assembled by polypeptides from F and S respectively to form an octamer structure composed of vestibule and beta-barrel structures; the vestibule and the connecting area of the beta-barrel-shaped structure are contraction areas; constructing a gamma-hemolysin nano pore canal on the phospholipid layer to enable liquid between the cis-type detection cell and the trans-type detection cell to be communicated; applying a voltage to obtain an opening current; adding a nucleic acid single molecule aqueous solution into the cis-detection pool, and applying voltage to enable the nucleic acid single molecule to pass through the contraction zone from the cis-terminal fragment of the gamma-hemolysin nano pore canal; measuring a blocking current through the gamma-hemolysin nanopore; identifying the nucleic acid single molecules according to the measured blocking current, and obtaining fragment sequencing information;
the nucleic acid single molecule is a target sequence and an identification code corresponding to the target sequence, 2 sections of complementary hybrid molecules are formed, and the nucleic acid single molecule is of an A-type double-helix structure, and the size of the A-type double-helix structure is structurally matched with the size of a contraction zone;
the target sequence is a first DNA, and the identification code corresponding to the first DNA is a second RNA;
the nucleotide sequence of the first DNA is shown in SEQ ID NO.10: the nucleotide sequence of the second RNA is shown as SEQ ID NO.11 and SEQ ID NO.12, and the amino acid sequence of the F-class polypeptide is shown as SEQ ID NO. 4; the amino acid sequence of the S-type polypeptide is shown as SEQ ID NO. 8; the method is a non-disease diagnostic or therapeutic method.
2. The method according to claim 1, characterized in that the salt solution has a concentration of 2-4M and the solvent of the salt solution is HOAc/KOAc buffer with ph=5.0.
3. The method according to claim 1 or 2, characterized in that the salt is potassium chloride or sodium chloride.
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|---|
| Cherie Shuo Tan.Detection of DNA Base Modifications by Biological Nanopores.University of Utah Theses & Dissertations.2018,第四章和第第5.2.3小节. * |
| Detection of DNA Base Modifications by Biological Nanopores;Cherie Shuo Tan;University of Utah Theses & Dissertations;第四章和第第5.2.3小节 * |
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